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Abstract

Changes in cardiac sympathetic innervation density in the developing and diseased heart have been implicated in the long-term regulation of cardiomyocyte function. Direct confirmation of this hypothesis requires the ability to quantitatively assess sympathetic nerve density in living heart. Accordingly, we generated a transgenic mouse line expressing enhanced green fluorescent protein (EGFP) under control of the sympathetic neuron-specific human dopamine-β-hydroxylase (hDβ H) gene promoter. Quantitative colocalization analyses of confocal images obtained from hDβ H-EGFP heart sections stained with an antibody to tyrosine hydroxylase (TH, a specific marker of sympathetic nerves) confirmed that the pattern of EGFP fluorescence mirrored that of anti-TH immunoreactivity, indicating that EGFP expression is restricted to sympathetic nerves. Next, we used two-photon fluorescence microscopy to visualize EGFP-expressing nerves in left ventricular epicardial layers of isolated perfused, adult hDβ H-EGFP hearts. Series of X-Y scans (105 × 105 μm2) were taken at 0.5 μm axial steps. Panels A and B show EGFP-expressing nerve segments in two X-Y scans obtained at 10 and 50 μm, respectively, below the surface. Volume rendering of the entire image stack (Panel C) provided a 3-D visualization of sympathetic nerves within the imaged tissue volume (105 × 105 × 50 μm3). Collectively, our results indicate that the hDβ H-EGFP transgenic mouse model is suitable for live imaging of the cardiac sympathetic neural network with microscopic resolution, enabling quantitative evaluation of innervation patterning and its role in regulating cardiomyocyte function in normal and diseased heart.